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Transcript of Acinetobacter oleivorans
Plasmid-Encoded Tetracycline Efflux Pump Protein AltersBacterial Stress Responses and Ecological Fitness ofAcinetobacter oleivoransHyerim Hong, Jaejoon Jung, Woojun Park*
Department of Environmental Science and Ecological Engineering, Korea University, Seoul, Republic of Korea
Abstract
Acquisition of the extracellular tetracycline (TC) resistance plasmid pAST2 affected host gene expression and phenotype inthe oil-degrading soil bacterium, Acinetobacter oleivorans DR1. Whole-transcriptome profiling of DR1 cells harboring pAST2revealed that all the plasmid genes were highly expressed under TC conditions, and the expression levels of many hostchromosomal genes were modulated by the presence of pAST2. The host energy burden imposed by replication of pAST2led to (i) lowered ATP concentrations, (ii) downregulated expression of many genes involved in cellular growth, and (iii)reduced growth rate. Interestingly, some phenotypes were restored by deleting the plasmid-encoded efflux pump genetetH, suggesting that the membrane integrity changes resulting from the incorporation of efflux pump proteins alsoresulted in altered host response under the tested conditions. Alteration of membrane integrity by tetH deletion was shownby measuring permeability of fluorescent probe and membrane hydrophobicity. The presence of the plasmid conferredperoxide and superoxide resistance to cells, but only peroxide resistance was diminished by tetH gene deletion, suggestingthat the plasmid-encoded membrane-bound efflux pump protein provided peroxide resistance. The downregulation offimbriae-related genes presumably led to reduced swimming motility, but this phenotype was recovered by tetH genedeletion. Our data suggest that not only the plasmid replication burden, but also its encoded efflux pump protein alteredhost chromosomal gene expression and phenotype, which also alters the ecological fitness of the host in the environment.
Citation: Hong H, Jung J, Park W (2014) Plasmid-Encoded Tetracycline Efflux Pump Protein Alters Bacterial Stress Responses and Ecological Fitness ofAcinetobacter oleivorans. PLoS ONE 9(9): e107716. doi:10.1371/journal.pone.0107716
Editor: Nancy E. Freitag, University of Illinois at Chicago College of Medicine, United States of America
Received May 23, 2014; Accepted August 21, 2014; Published September 17, 2014
Copyright: � 2014 Hong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The RNA-Seq data were deposited in theNational Center for Biotechnology Information (NCBI) GEO site under accession numbers GSE38340, GSE44428, and GSE55239.
Funding: This work was supported by the Mid-career Researcher Program through an NRF grant (2014R1A2A2A05007010) funded by the Ministry of Science, ICT& Future Planning (MSIP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: [email protected]
Introduction
Plasmids carrying antibiotic resistance genes are one of the
primary sources of multidrug resistance in pathogens [1,2].
Plasmids can be transferred to microorganisms through horizontal
gene transfer (HGT), by methods such as conjugation and
transformation, which can occur inside the hosts, as well as in
the environment [3–5]. Many conjugative plasmids harboring
antibiotic resistance genes have been shown to be present in
wastewater treatment plants (WWTP) [6–10]. Antibiotics, includ-
ing tetracycline (TC), could exert selective pressure on these
plasmids and facilitate the spread of antibiotic resistance genes in
the environment [11–13]. TC prevents bacterial growth by
binding to a single site on the 30S ribosomal subunit, and
preventing attachment of aminoacyl tRNA molecules to the
ribosome [14]. TC-related antibiotics have been applied in clinics,
and in agricultural and aquatic settings for disease control and
animal growth promotion, and are frequently detected in the
effluent from WWTPs [15–17]. TC resistance could be acquired
by three known mechanisms: energy-driven efflux pumps,
ribosomal protection proteins, and TC-modifying enzymes [18].
Efflux pumps encoded by several genes, such as tetA, tetC, tetE,tetG, and tetH, have been reported to be a major mechanism of
TC resistance; these genes are frequently located in plasmids and
transposons [19–21].
Antibiotic resistance is known to exert a metabolic cost on
bacteria, and antibiotic-resistant cells often show a reduced growth
rate [22,23]. The correlation between fitness cost and antibiotic
resistance has been extensively reviewed [24–26]. Previously, we
demonstrated that not only bacterial growth, but also many
cellular processes including quorum sensing, motility, and stress
response could be affected by acquiring antibiotic resistance
[27,28]. A newly described tetracycline resistance plasmid,
pAST2, was isolated from an activated sludge, and its entire
plasmid genome was characterized [29]. The pAST2 plasmid
encodes the TC efflux pump, tetH, and tetR genes that confer
tetracycline resistance to the host. Expression of the tetH gene is
known to be regulated by a repressor, TetR [30,31]. Based on our
bioinformatics study, this tetH-tetR module originated in bovine
and swine pathogens such as Pasteurella multocida, Mannheimiahaemolytica, and Actinobacillus pleuropneumoniae [32,33]. Re-
cently, we demonstrated that the acquisition of this plasmid alters
the phenotypic characteristics of the oil-degrading microbe
Acinetobacter oleivorans DR1 [29]. Presence of the plasmid
incurred high ecological costs for phenotypic and physiological
PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e107716
functions in A. oleivorans DR1. This observation is consistent with
the fact that antibiotic resistance plasmids alter the expression of
host chromosomal genes and ecological adaptation in Salmonella[34].
The aims of this study were (i) to gain insight into the link
between changes in host chromosomal expression and phenotypic
changes in the presence of the pAST2 plasmid, and (ii) to
distinguish the metabolic costs incurred by the plasmid replication
burden and by changes in membrane integrity caused by addition
of the plasmid-encoded tetracycline efflux pump. We compared
four RNA-Seq transcriptomes of the wild-type and plasmid-
harboring cells in the presence and absence of TC. To understand
the fitness costs of the efflux pump, a tetH-tetR knockout plasmid
was generated and tested under different environmental condi-
tions. Our findings provide evidence that the expression of many
host chromosomal genes can be modulated by a foreign TC
resistance plasmid, altering the host’s biological fitness. We also
confirm that the plasmid-encoded efflux pump protein alone can
influence host phenotype by affecting membrane integrity and
permeability.
Results
1. RNA-Seq-based transcriptional profiling of A. oleivoransDR1 harboring pAST2
To identify altered expression of genes on the host chromosome
in the presence of the plasmid, we conducted RNA-Seq whole-
transcriptome analysis using DR1 cells harboring pAST2 in the
presence and absence of TC. TC was applied to each exponen-
tially grown cell at a minimal inhibitory concentration (MIC,
1 mg/ml) for 15 min. Fifteen min was determined to be an optimal
incubation time so as not to cause serious cell-death by tetracycline
in liquid culture. RNA-Seq analyses were performed with wild
type DR1, and wild type carrying pAST2, TC-treated DR1, and
TC-treated DR1 (pAST2). They were designated as DR1, DR1
(pAST2), DR1-TC, and DR1 (pAST2)-TC, respectively. The total
number of reads from DR1, DR1 (pAST2), DR1-TC, and DR1
(pAST2)-TC was 6583302, 36127802, 17509940, and 20507109,
respectively. The sequencing coverage for each sample was 57,
868, 151, and 177-fold, respectively. More than 99% of the reads
were mapped to the reference genome sequence, which indicates
satisfactory sequencing quality. Overall gene expression profiles
indicated that dramatic down-regulation occurred in the presence
of pAST2 (Figure S1A). With the plasmid, a total of 275 genes
were upregulated, and 2125 genes were downregulated (Table 1).
However, the majority of genes were upregulated in DR1 cells
with pAST2 in the presence of TC (Figure S1B, S1C). This level of
upregulation was not observed in the absence of the plasmid
(Figure S1D, Table 1).
DR1 cells harboring pAST2 showed that transcription-related
(COG K) and signal transduction (COG T) genes were
downregulated (Figure S2A, Table 1). In contrast, the presence
of the plasmid under TC conditions increased expression of many
genes in categories F (nucleotide metabolism and transport) and K
(transcription), whereas a number of genes in categories A (RNA
processing and modification), J (translation), and N (motility-
related) were downregulated (Figure S2B, S2C). The wild-type
strain under TC repressed the expression of cell cycle control
(COG D), nucleotide metabolism and transport (COG F), cell wall
structure and outer membrane (COG M), and coenzyme
metabolism (COG H) gene expression (Figure S2D). Our data
show that the presence of the plasmid could modulate host
chromosomal expression.
All the genes on the plasmid appeared to be actively
upregulated by more than 2 fold under TC conditions (Table 2).
Consistent with our previous observation that the tetH promoter
was strongly induced at sub-MIC concentrations (0.5 mg/ml) of
TC [29], the tetH gene was the most strongly upregulated gene (35
fold). Interestingly, mobilization protein A showed the second
highest increase in expression (29 fold) under TC conditions,
which suggested that TC could facilitate plasmid transfer in the
environment.
2. Plasmid-mediated tetracycline resistance consumesintracellular energy in bacterial cells
Because our transcriptomic data showed that many RNA
polymerase-related genes were significantly downregulated in
DR1 (pAST2), we hypothesized that presence of the plasmid
imposed an energy burden on the host (Table 3). However,
addition of TC reversed this tendency, indicating that cells
harboring the plasmid actively transcribed many defense genes
under TC condition. ATP-related genes were also down-regulated
in the presence of the plasmid, which could not be seen under TC
(Table 3). Reduction of ATP concentration could be expected
with pAST2. However, no clear pattern was observed in the
presence of TC. To test this, growth rate and intracellular ATP
concentrations were measured with or without TC (Figure 1).
Growth rate was significantly reduced in the presence of the
plasmid regardless of the presence of the tetH gene, which
suggested that the replication burden was the major cause of the
growth defect (Figure 1A). However, the degree of growth defect
differed in the presence and absence of the tetH gene, which
indicated that expression and membrane incorporation of the
plasmid-encoded efflux pump imposed a metabolic cost on the
host. As expected, the wild-type strain with pAST2 gained a
Table 1. RNA-Seq-based transcriptional profiling.
Fold change
DR1(pAST2)/DR1DR1(pAST2)-TC/DR1-TC
DR1(pAST2)-TC/DR1(pAST2) DR1-TC/DR1
Total upregulated genes 275 1222 2492 713
Total downregulated genes 2125 284 115 881
COG category containing the highestproportion of upregulated genes
– F K N
COG category containing the highestproportion of downregulated genes
K, T A, J N D, F, H, M
doi:10.1371/journal.pone.0107716.t001
Alteration of Host Phenotypes by Antibiotic Resistance Plasmid
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growth advantage under TC conditions and deletion of the tetHgene from the plasmid decreased growth under TC conditions
(Figure 1A). Accordingly, wild-type DR1 cells had lower ATP
levels in the presence of TC (Figure 1B). ATP concentrations were
significantly lower with the plasmid regardless of TC presence. It is
interesting to note that under TC conditions, plasmid-carrying
cells appeared to use energy efficiently for boosting their growth
rate, because their growth rate was very high in spite of low ATP
levels (Figure 1A, 1B). A further reduction in ATP levels was
measured when the tetH gene was deleted, compared to plasmid-
carrying cells (Figure 1B). Consistent with the growth rate
measurements, the addition of the efflux pump appeared to
interfere with cellular processes, resulting in lower ATP levels and
decreased growth.
3. Loss of hexadecane degradation ability in strain DR1harboring pAST2
Acinetobacter is well-known for its ability to degrade alkanes
[35–37]. It has been shown that the hexadecane degradation
pathway is present in the A. oleivorans DR1 genome [38–41].
Many genes involved in hexadecane utilization, including alkane
1-monooxygenase encoded by alkB (AOLE_10550) and aldehyde
dehydrogenase encoded by putA (AOLE_06655) were downreg-
ulated in the presence of the plasmid (Table 3). However, under
TC conditions, alkB gene and Zn-dependent aldehyde dehydro-
genase (adhC) were upregulated, indicating that different alkane
degradation pathways might be used under different conditions.
With hexadecane as the sole carbon source and an absence of TC,
the growth rates of all he pAST2-harboring cells were lower than
those of the wild-type cells (Figure 2A). Both DR1 (pAST2) and
DR1 (pAST2DPtetH) showed better utilization of hexadecane than
wild-type cells in the presence of TC. However, deletion of the
tetH gene did not affect growth rate on hexadecane, which
suggests that alteration of membrane integrity by TetH is not an
important factor for hexadecane utilization. A microbial adhesion
to hydrocarbon (MATH) assay was conducted to determine
membrane hydrophobicity. MATH values were lower in the
presence of pAST2. Thus, deletion of the tetH gene altered
membrane hydrophobicity (Figure 2B).
4. Antibiotic sensitivity and response to oxidative stressin DR1 strains harboring pAST2
Our RNA-Seq analysis showed that many multidrug-efflux
pump-related genes were downregulated in the absence of TC
(Table S1). Expression of many chromosomal efflux pump genes
was repressed in the presence of pAST2. Aminoglycoside
resistance efflux pump (AOLE_02025), and two beta-lactamase
genes (AOLE_05220 and AOLE_11070) were downregulated
11.9, 11,7 and 7.0 fold, respectively. The pAST2 plasmid appears
to elevate expression level of many chromosomal genes involved in
stress resistance under TC-amended condition (Table S1). Various
chemical compounds were applied to the DR1 wild-type strains
and plasmid-harboring strains to determine the effects of the
exogenous plasmid on host response to various stresses. Antibiotics
were amended at sub-MIC levels. Interestingly, the DR1 strain
harboring the plasmid became sensitive to ampicillin in the
absence of TC (Figure S3A). This sensitivity was lost when the
tetH gene was deleted, which showed that TetH is critical for
ampicillin sensitivity. Kanamycin gene cassette replacing the tetHgene in the plasmid was responsible for kanamycin resistance.
Under TC conditions (Figure S3B), the DR1 strain harboring the
plasmid appeared to have greater resistance to several antibiotics,
with the exception of ampicillin. Cell-wall inhibition by ampicillin
thus appears to be strongly affected by the presence of the TetH
efflux pump. Our data showed that the plasmid replication burden
altered genetic and phenotypic responses of the host to antibiotics;
the tetH-encoded efflux pump alone could affect these host
responses.
Several genes functioning in oxidative stress defense were
upregulated in the presence of the plasmid (thioredoxin, 3.6 fold;
glutaredoxin, 2.6 fold; catalase, katG, 2.4 fold; catalase, katE, 2.0
fold; rubredoxin reductase, nirB, 1.8 fold) although some oxidative
Table 2. Gene expression (fold change) values of pAST2 under tetracycline treatment conditions in A. oleivorans DR1.
Gene/orf Position Product RPKM Fold change
DR1(pAST2)-TC DR1(pAST2) pAST2-TC/pAST2
repB 71–1063 plasmid replication protein B 493.66 127.81 3.86
tnp 1185–2069 transposase 1470.09 768.56 1.91
tetH 2410–3612 major facilitator transporter 431.35 12.18 35.41
tetR 3704–4327 tetracycline repressor protein 814.01 161.14 5.05
orf1 4330–5274 hypothetical protein 465.42 21.75 21.40
orf2 5274–5876 hypothetical protein 426.90 15.44 27.65
orf3 6071–6595 conserved hypothetical protein 326.26 44.77 7.29
orf4 6670–7065 predicted protein 346.53 39.33 8.81
orf5 7040–7918 predicted protein 299.74 43.93 6.82
cueR/merR 8005–8370 Cu(I)-responsive transcriptional regulator 273.41 42.31 6.46
orf6 8631–9443 hypothetical protein 502.21 71.42 7.03
orf7 9535–9864 transcriptional regulator, XRE family 601.36 163.10 3.69
orf8 9855–10184 helix-turn-helix domain protein 330.11 43.01 7.68
mobA 10385–10543 mobilization protein A 151.37 5.22 29.00
mobC 10533–10898 mobilization protein C 285.72 18.38 15.55
orf9 11231–11818 hypothetical protein 305.42 47.02 6.50
doi:10.1371/journal.pone.0107716.t002
Alteration of Host Phenotypes by Antibiotic Resistance Plasmid
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stress-related genes were downregulated (Table S1). The oxidative
stress responses of the host harboring the plasmid were tested using
compounds such as superoxide [menadione (MD), and paraquat
(PQ)] and hydrogen peroxide [hydroperoxide (H2O2), cumene
hydroperoxide (CHP)]. The results showed that DR1 harboring
pAST2 had greater resistance to all oxidative stress compounds
than wild-type cells and tetH-deleted strains (Figure 3A and 3B).
DR1 strains, except cells harboring pAST2, were more sensitive to
peroxide compounds, which increased the size of the inhibition
zone (Figure 3A). Under TC conditions, plasmid-harboring DR1
strains displayed a consistent radius with control, and the wild-type
strain was more sensitive to oxidative stress compounds (Fig-
ure 3B). Our data demonstrated that the pAST2 plasmid can
increase the expression of many stress response-associated genes,
reducing sensitivity to oxidative and antibiotic stresses. Moreover,
our data provide evidence that the plasmid-mediated efflux pump
protein conferred peroxide resistance.
5. Additional phenotypic changes caused by the plasmid-encoded TetH efflux pump - motility, membranepermeability, and morphology of fimbria appendages
RNA-Seq data showed that expression of many outer mem-
brane proteins (Table S2) and fimbriae/pili-related genes was
altered (Table 4). Decreased expression of fimbrial proteins
encoded by fim genes was consistent with poor motility and
decreased surface attachment. [fimA (AOLE_09780), fimC(AOLE_06715), and fimT (AOLE_03010) were downregulated
by 7.0, 5.7, and 4.8 fold, respectively (Table 4)]. Swarming
motility of wild-type cells showed a distinct branched structure
with serrated edges on semi-solid nutrient agar (Figure 4A).
Figure 1. Determination of bacterial growth rates and ATP concentrations in DR1 strains. (A) Growth of DR1 cells in nutrient mediacontaining tetracycline. Cells were grown at 30uC in nutrient media supplemented with or without TC (sub-MIC concentration). The growth rate ofeach strain was monitored by measuring the optical density (OD) of cultures at 600 nm. Error bars indicate the standard deviation of threeindependent experiments. (B) Influence of plasmid on ATP generation. The ATP concentration is expressed as molar concentration per mg of protein.All data show the average of three replicates, and one standard deviation is shown. Statistical analyses were conducted using Student’s t-test. A letteron the bar graph indicates the level of significance. Bars denoted by the same letter are not statistically significant (p.0.05).doi:10.1371/journal.pone.0107716.g001
Alteration of Host Phenotypes by Antibiotic Resistance Plasmid
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However, swarming motility of strain DR1 (pAST2) exhibited a
mucoid phenotype with irregular form. Interestingly, the tetHmutant strain maintained a branched form similar to the wild-type
strain. Under TC conditions, the spreading zones of the three
DR1 strains showed highly mucoid phenotypes with exopolysac-
charide (EPS) overproduction. Interestingly, cells harboring
pAST2 appeared to have reduced swimming motility, but the
tetH mutant strain showed recovered swimming motility (Fig-
ure 4B). On TC-containing swimming agar plates, motility of the
tetH mutant and wild-type strains was equally reduced.
Membrane permeability was measured using 8-anilino-1-
naphthylenesulfonic acid (ANS), a neutrally charged, hydrophobic
probe that exhibits enhanced fluorescence in membrane-damaged
cells [42,43]. Membrane permeability is shown as red fluorescence
intensity penetrating the cell membrane divided by mg protein
(Figure 4C). Similar membrane permeability was observed in both
wild-type and the tetH mutant strains. In contrast, the DR1
(pAST2) strain showed reduced permeability, presumably caused
by cell membrane alteration by the TC resistant efflux pump.
Under TC conditions, no significant differences were observed,
although TetH deletion caused a slight increase in membrane
permeability.
Morphology of bacterial surface appendages was observed using
transmission electron microscopy (TEM) (Figure 5). A. oleivoransDR1 cells have been reported to possess two major fimbrial
appendages on the outer membrane [28,41]. Thick fimbriae
(28.2–29.3 nm in diameter) were sparsely distributed around the
periphery of DR1 wild-type cells, and thin fimbriae (6.8–10 nm in
diameter) were densely present on the membrane surface.
Distribution of both types of fimbriae on the tetH mutant strain
closely resembles that of wild-type cells; on DR1 cells harboring
pAST2, thin fimbriae were absent but the thick fimbriae
remained. Under TC conditions, the thick fimbriae were absent
on DR1 (pAST2) cells, and only the thinner structures were
observed. In addition, the presence of the pAST2 plasmid
encoding the efflux pump increased membrane thickness from
50 nm to 60 nm in the absence of TC. However, a high level of
membrane destruction was observed in both the pAST2-harboring
strain and the tetH mutant strain.
6. Fitness cost of plasmid-mediated tetracyclineresistance
A competition assay between the wild-type and plasmid-
harboring strains was performed to measure fitness; growth was
measured in nutrient media with or without antibiotics and
oxidative compounds at sub-MIC concentrations (Figure 6). MSB
medium was amended with 2% n-hexadecane to determine the
competitive utilization of hexadecane. Relative fitness was
expressed as the proportion with DR1 (pAST2) in co-culture
cells. We found that the plasmid-harboring strain showed low
fitness under non-antibiotic selective pressure (Figure 6A). This
result was accordance with our observation that the growth rate of
DR1 (pAST2) was much lower than that of the wild-type without
TC. Interestingly, when norfloxacin was applied to the nutrient
media, the plasmid-harboring strain possessed their proportion
with equal to wild-type strain (0.9860.27). Hydrogen peroxide
(sub-MIC) applied in co-culture appeared to be uncompetitive
(0.1160.06). In contrast to this observation, peroxide stress at an
above-MIC concentration provided resistance to the pAST2-
carrying DR1 strain (Figure 3A). The plasmid-harboring strain
Table 3. Gene expression (fold change) values of genes related to transcription, ATPase, ATP synthase, and hexadecanedegradation.
Locus_tag Gene Product Fold change
DR1(pAST2)/DR1 DR1(pAST2)-TC/DR1-TC
Transcription-related genes
AOLE_16340 rpoN RNA polymerase factor sigma-54 27.19 4.57
AOLE_04330 rpoD RNA polymerase factor sigma-70 26.63 1.68
AOLE_12520 – DNA-directed RNA polymerase 25.25 1.24
AOLE_12655 rpoE RNA polymerase sigma-24 subunit 23.04 1.64
AOLE_05735 rpoH RNA polymerase factor sigma-32 23.00 1.28
AOLE_08705 rocR sigma54 specific transcriptional regulator 22.00 2.00
ATPase and ATP synthase-related genes
AOLE_17825 recN ATPase 29.21 1.91
AOLE_18605 atpI ATP synthase I chain family protein 25.33 22.75
AOLE_15995 – putative ATPase 24.80 1.87
AOLE_08275 clpS ATP-dependent Clp protease adaptor protein 22.51 1.07
AOLE_18600 atpB F0F1 ATP synthase subunit A 21.70 1.09
Hexadecane degradation-related genes
AOLE_06655 putA NAD-dependent aldehyde dehydrogenase 22.79 27.41
AOLE_10550 alkB alkane 1-monooxygenase 21.58 2.41
AOLE_14590 nirB 3-phenylpropionate dioxygenase ferredoxin 21.46 1.30
AOLE_03630 eutG alcohol dehydrogenase 1.37 1.77
AOLE_09790 adhC Zn-dependent alcohol dehydrogenase 1.60 4.11
AOLE_14370 nirB rubredoxin-NAD(+) reductase 1.80 2.00
doi:10.1371/journal.pone.0107716.t003
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showed 2.4 fold (MSB media) and 6 fold (nutrient broth) higher
relative fitness in the presence of TC (Figure 6B). Our results
suggest that plasmid-mediated TC resistance mitigated fitness costs
in the presence of TC, whereas the plasmid-carrying population
was considerably reduced under antibiotic-free conditions.
Discussion
Bacteria gain antibiotic resistance by acquiring a plasmid
encoding antibiotic resistance genes, but cells harboring the
plasmid experience loss of fitness in the absence of antibiotic
selective pressure [44–46]. Research on the influence of plasmid-
mediated antibiotic resistance on bacterial physiology and fitness
costs have primarily focused on growth defect [47–51]. Carriage of
a plasmid yields clear benefits when the corresponding antibiotic is
present [48,52]. The fitness reduction observed in plasmid-
carrying bacteria may contribute to the instability of plasmids in
the environment because of competition with plasmid-free
bacteria [53]. However, other studies have shown that expression
of plasmid-encoded antibiotic resistance genes has an adverse
effect on the reproductive fitness of plasmid-containing bacteria
[54] and that harboring an antibiotic resistance plasmid triggered
transcriptional deregulation [34]. Use of molecular machinery and
energy for expressing plasmid genes in a host presumably alters
expression of host genes. Thus, many other phenotypes may be
modulated by possession of a plasmid. Elimination of resistance
genes from a plasmid can lower the fitness burden on host bacteria
[55,56]. In addition, repression of a resistance gene can be
effective in avoiding the cost of resistance in an antibiotic-free
environment [57].
Acinetobacter species carrying a resistance plasmid showed
decreased fitness in the absence of antibiotics [58]. We have also
shown a relationship between bacterial fitness costs and antibiotic
Figure 2. Hexadecane degradation and microbial adherence to n-hexadecane (MATH). (A) Hexadecane degradation rate (min21).Utilization of hexadecane was monitored by measuring the OD600. Error bars indicate the standard deviation of three independent experiments.Statistical significance of differences was calculated using Student’s t-test. (B) Microbial adhesion to hydrocarbon (MATH) assay. All samples wereanalyzed in triplicate. Statistical analyses were conducted using Student’s t-test. A letter on the bar graph indicates the level of significance. Barsdenoted by the same letter are not statistically significant (p.0.05).doi:10.1371/journal.pone.0107716.g002
Alteration of Host Phenotypes by Antibiotic Resistance Plasmid
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resistance in Acinetobacter oleivorans DR1 [27,40]. Acquisition of
the extracellular plasmid pAST2 altered phenotypes, and the
phenotypic changes are thought to be linked to changes in host
gene expression [29]. In order to gain further insight into the
phenotypic changes caused by uptake of extracellular genetic
material, we performed an RNA-Seq analysis of the entire
transcriptome. To validate our RNA-Seq result, quantitative
real-time PCR (qRT-PCR) confirmed the gene expression of 10
genes selected based on category and expression value (Figure S4).
The results showed that the expression values of those genes were
closely matched to RNA-Seq data (Figure S4). Our findings
showed that all plasmid-encoded genes were highly expressed,
which altered not only host gene expression, but caused
phenotypic and physiological changes. Interestingly, our tran-
scriptomic data showed that many membrane-related genes and
most fimbrial proteins encoded by fim genes were considerably
downregulated (Table 4, Table S2). In contrast, membrane
appendage pilin-related genes were highly upregulated [e.g. pilA(AOLE_01545, 218.7-fold increase), data not shown] by posses-
sion of the plasmid. However, we did not observe pili in our TEM
analysis (Figure 5). We are unable to explain this discrepancy, but
hypothesize that the pilus may be lost during preparation of TEM
images. It is interesting to note that the natural competence-
associated type IV pilus assembly protein encoded by
AOLE_15230 (3.5-fold increase) and AOLE_17785 (3.69-fold
increase) were also upregulated, and plasmid-mediated horizontal
gene transfer by expression of pilus genes merits investigation in
this strain. The low levels of fimbriae expression observed may be
linked to loss of adhesion to hexadecane droplets, reduced
membrane permeability, or diminished motility [40].
Membrane integrity and permeability may play important roles
in many bacterial stress responses [59,60]. Our data demonstrated
for the first time that, in addition to the plasmid replication
burden, the plasmid-encoded membrane bound efflux pump
encoded by tetH gene is important for altering bacterial physiology
and phenotypes such as peroxide resistance, membrane perme-
ability, and fimbria expression. Physical incorporation of the efflux
pump into the membrane appears to be critical for altering
membrane integrity. The efflux pump protein encoded by TetH in
pAST2 contains 400 amino acids with an unusual 11 transmem-
brane domains; most tetracycline efflux pumps, such as TetA,
contain 12 transmembrane helices (data not shown). The TetH
Figure 3. Sensitivity of Acinetobacter strains to oxidative stress. (A) Oxidative stress inhibition zone assay in nutrient media (controlcondition). (B) Oxidative stress inhibition zone assay with TC. Oxidative stress compounds, including superoxide (menadione, MD; paraquat, PQ) andhydrogen peroxide (hydroperoxide, H2O2; cumene hydroperoxide, CHP), were tested on exponentially grown cells.doi:10.1371/journal.pone.0107716.g003
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protein belongs to the major facilitator superfamily (MFS) of
transporters, which has little specificity [61]. In many bacterial
genomes, membrane-transport proteins comprise only 0.1–1.0%
of the total proteins, and the expression of many membrane
proteins is very low (less than 0.1% of total cell proteins) under
typical conditions [62]. Our data demonstrated that high
expression of the plasmid-encoded TetH altered the physiological
status of cells, including hydrogen peroxide resistance, reduction of
motility, and altered fimbria expression. Modulation of biological
fitness by the plasmid-mediated tetracycline resistance TetH efflux
pump may be attributed to the interference of the plasmid-
encoded membrane-bound tetracycline efflux pump. In conclu-
sion, the significant alteration of bacterial membrane integrity
caused by the plasmid-mediated efflux pump affects bacterial
phenotype and biological fitness in the environment.
Materials and Methods
1. Bacterial strains and growth conditionsThe bacterial strains, plasmids, and primers (from 59 to 39)
utilized in this study are listed in Table S3. The hexadecane-
degrading bacterium, Acinetobacter oleivorans DR1, was grown at
30uC in nutrient broth (Marine BioProducts, Canada) and shaken
at 220 rpm for aeration. DR1 strains harboring plasmids were
grown under the same conditions as the wild-type DR1 strain. The
E. coli strains were grown at 37uC in LB broth and shaken at
220 rpm for aeration. The tetracycline-resistance plasmid pAST2
that was transformed into strain DR1 used in this study was
environmentally isolated, as described previously [29]. The
complete sequence of the plasmid was deposited in the National
Center for Biotechnology Information (NCBI) database under
accession number KC734561. The genome sequence of A.oleivorans DR1 was retrieved from the NCBI database (accession
number NC_014259.1).
2. RNA-Seq analysisStrain DR1 carrying pAST2 and wild-type strain DR1 were
grown overnight at 30uC with shaking at 220 rpm. A 50 ml
stationary-phase culture was used to inoculate 5 ml fresh nutrient
medium and then incubated under the same conditions. Each
strain was grown to the exponential phase (OD600 of ,0.4) at
30uC in nutrient medium supplemented with or without 1 mg/ml
tetracycline over a period of 15 min. Total RNA was extracted
using an RNeasy Mini Kit (Qiagen, USA), following the
manufacturer’s instructions. All procedures for RNA sequencing
and alignment were carried out by Chunlab (Seoul, South Korea).
RNA was subjected to a subtractive Hyb-based rRNA removal
process using the MICROBExpress Bacterial mRNA Enrichment
Kit (Ambion, USA). Subsequent processing, including library
construction, was conducted as described previously [63]. RNA
sequencing was performed using two runs of the Illumina Genome
Analyzer IIx to generate single-ended 36-bp reads. Quality-filtered
reads were aligned to the A. oleivorans DR1 genome as a
reference sequence by using the CLC Genomics Workbench 4.0
(CLC Bio, Denmark). Mapping was based on a minimal length of
32 bp with an allowance of up to 2 mismatches. The relative
transcript abundance was measured in reads per kilobase of exon
sequence per million mapped sequence reads (RPKM) [64]. The
mapping results were visualized using the CLRNA-Seq programs
(Chunlab, South Korea). The fold-change [RPKM of DR1
(pAST2)/RPKM of DR1, RPKM of DR1-TC/RPKM of DR1,
RPKM of DR1 (pAST2)-TC/RPKM of DR1 (pAST2), and
RPKM of DR1 (pAST2)-TC/RPKM of DR1-TC] represented
the level of gene up- or downregulation. Genes showing fold-
changes larger than 2.0 were designated as upregulated, and those
showing fold-changes greater than 22.0 were designated as
downregulated. The RNA-Seq data were deposited in the
National Center for Biotechnology Information (NCBI) GEO site
under accession numbers GSE38340, GSE44428, and GSE55239.
Figure 4. Bacterial capsule-related phenotype test. The plasmid-encoded membrane bound TC efflux pump alters bacterial outermembrane and capsule related appendages. (A) Swarming motility(0.5%), (B) Swimming motility (0.2%), (C) Membrane permeability assay.All quantitative data were obtained in triplicate. A letter on the bargraph indicates the level of significance. Bars denoted by the sameletter are not statistically significant (p.0.05).doi:10.1371/journal.pone.0107716.g004
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PLOS ONE | www.plosone.org 8 September 2014 | Volume 9 | Issue 9 | e107716
3. Gene expression analysis using qRT-PCRWild-type DR1 and DR1(pAST2) were grown to the exponen-
tial growth phase (OD600 = ,0.4) at 30uC in nutrient medium.
The exponentially growing cells (OD600 = ,0.4) were treated with
1 mg/ml TC and then incubated for 15 min. Total RNA was
extracted from 5 ml of cultures using an RNeasy Mini kit (Qiagen,
Valencia, CA, USA) according to the manufacturer’s instructions.
cDNA was synthesized from 1 mg RNA by using gene specific
primers based on sequence of A. oleivorans genome (Table S3)
and the primers for genes were used as templates for quantitative
real-time PCR (qRT-PCR). The 25 ml PCR mixture included
12.5 ml iQ SYBR Green Supermix (Bio-Rad), 2 ml of each primer
Table 4. Expression profile of bacterial fimbriae-related genes.
Locus_tag Gene Product Fold change
DR1(pAST2)/DR1 DR1(pAST2)-TC/DR1-TC
AOLE_09780 fimA fimbrial family protein 27.01 1.02
AOLE_06715 fimC fimbrial protein 25.69 21.42
AOLE_09775 fimD fimbrial protein 25.66 1.51
AOLE_06720 fimD fimbrial protein 25.38 21.80
AOLE_09770 fimC chaperone protein mrkB 24.94 21.01
AOLE_03010 fimT type IV fimbrial biogenesis protein FimT 24.80 24.19
AOLE_07050 fimD fimbrial protein 24.06 2.13
AOLE_16925 pilF stability protein 23.95 3.05
AOLE_11120 fimC fimbrial chaperone 23.29 21.17
AOLE_17295 fimV FimV domain-containing protein 22.53 21.15
AOLE_11125 fimD fimbrial protein 22.21 1.25
doi:10.1371/journal.pone.0107716.t004
Figure 5. TEM analysis of DR1 strains. Microscopic images were obtained by transmission electron microscopy (TEM). Cells were cut into 70-nm-thick sections and double stained with uranyl acetate and lead citrate for contrast staining. The cross-section of each specimen is displayed in a smallbox alongside the longitudinal section. Bar indicates 250 nm.doi:10.1371/journal.pone.0107716.g005
Alteration of Host Phenotypes by Antibiotic Resistance Plasmid
PLOS ONE | www.plosone.org 9 September 2014 | Volume 9 | Issue 9 | e107716
(0.5 mM), 3 ml cDNA, and 5.5 ml distilled water. The PCR
conditions were 95uC for 3 min, followed by 35 cycles consisting of
23 s at 95uC, 23 s at 60uC, and 23 s at 72uC. The expression level
of each gene was normalized to the 16S rRNA expression level
that was quantified with 16s rRNA-341F/16s rRNA-534R
primers. Relative quantifications were performed in triplicate.
4. Construction of the tetH-tetR mutant, DR1(pAST2DPtetH)
The suicide vector pCVD442 [65] was used to construct a tetH-tetR mutant. Polymerase chain reaction (PCR) and gel electro-
phoresis were carried out as described previously [66]. A 463-bp
fragment of the internal region of the tetH gene that had been
amplified using tetH-F/tetH-R primers was cloned into the KpnI/
BamHI cloning sites of the pBBR1MCS4 vector, generating
pBBR1MCS4-tetH (Figure S5). The plasmid was extracted using a
Dyne Plasmid Miniprep Kit (DYNEBIO, Korea). The amplicon
(440 bp) of the partial region of the tetR gene obtained using the
tetR-F/tetR-R primer pair was cloned into the BamHI/SacI
cloning sites of the pBBR1MCS4-tetH vector to yield
pBBR1MCS4-tetHtetR. A full-length kanamycin cassette
(1,264 bp) from the plasmid pUC4K was digested with BamHI
and cloned into the pBBR1MCS4-tetHtetR vector to obtain
pBBR1MCS4-tetHtetR::km. The constructed plasmid was digested
using KpnI/SacI (Thermo Fisher Scientific, USA) to obtain the
tetHtetR::km fragment and finally cloned into the pCVD442
vector to yield pCVD442-tetHtetR::km. The constructed plasmid
was then introduced into A. oleivorans DR1 (pAST2) by
electroporation. Transformation was performed with 2.5 ml of
plasmid DNA and 50 ml A. oleivorans DR1 (pAST2) competent
cells using a micropulser (Bio-Rad, USA) with a time constant
range of 4.0–5.0 ms and a constant voltage of 5.5–6 kV. PCR was
conducted to confirm the inserted fragments using the tetH-F/
tetR-R primer set (Table S3).
5. Bioluminescent ATP assayIntracellular ATP concentrations were measured using the
ENLITEN ATP Assay System Bioluminescence Detection Kit
(Promega, USA). Exponentially grown cells (OD600 of ,0.4) were
treated with 0.5 mg/ml TC or without TC for 15 min and cells
were harvested by centrifugation (13,0006g, 1 min). The pellet
was suspended with 1% trichloroacetic acid (TCA) buffer for ATP
extraction. Each sample in TCA buffer was diluted 5 fold with
Tris-acetate buffer to neutralize the extracts. The sample (20 ml)
was then mixed with 100 ml of luciferin–luciferase reagent, and the
bioluminescence intensity, in relative light units (RLU), was
recorded immediately using a microtiter plate reader (VICTOR3,
BioRad, USA) with 10 s integration time. An ATP standard curve
was constructed in accordance with the manufacturer’s instruc-
tions (Figure S6). The ATP concentration was calculated as molar
concentration per mg of protein. ATP assays were performed from
triplicate experiments. Statistical significance of differences was
calculated using Student’s t-test.
6. Hexadecane degradation and microbial adherence ton-hexadecane (MATH)
A. oleivorans DR1 strains were inoculated at ,106 CFU/ml in
20 ml of MSB media supplemented with 2% (v/v) n-hexadecane
(Sigma, USA). Degradation of hexadecane was monitored by
measuring the OD600 of each strain. After TC (0.5 mg/ml) was
added to the exponentially grown cells (OD600 of ,0.4) and they
were incubated for 30 min, 2 ml of bacterial cells were harvested
by centrifugation (13,0006g, 1 min) and washed twice with
phosphate-buffered saline (PBS). The OD600 of the resuspended
cells was measured in the same way as the initial OD. The cells
were agitated with 200 ml of n-hexadecane using a vortexer for
10 min. After 15 min of immobilization, the mixed solution was
divided into two separate phases. We measured the absorbance of
the aqueous layer at 600 nm as the final OD. The hydrophobicity
of each strain was calculated as the final OD divided by the initial
OD, expressed as a percentage (%). The quantitative data were
obtained from triplicate experiments. Statistical significance of
differences was calculated using Student’s t-test.
7. Sensitivity of Acinetobacter strains to antibioticsA. oleivorans DR1 strains were grown in nutrient broth with
shaking at 30uC. The stationary-grown cells were diluted 100 fold
in 5 ml of fresh media and incubated until the OD600 of the cells
reached the exponential phase (OD600 of ,0.4). The exponentially
growing cells were harvested by centrifugation (13,0006g, 1 min)
and washed twice with PBS. One hundred-microliter samples
Figure 6. Fitness cost of plasmid-mediated tetracycline resis-tance. (A) Fitness by long-term survival of wild-type DR1 and DR1(pAST2) in mixed cultures under test conditions. Norfloxacin andhydrogen peroxide were supplemented with sub-inhibitory concentra-tions in nutrient media. (B) Fitness under TC-treated conditions. Therelative fitness is expressed as the ratio of DR1 (pAST2) to wild-type(fitness value; 1.0). Quantitative data were obtained from twoindependent mixed cultures.doi:10.1371/journal.pone.0107716.g006
Alteration of Host Phenotypes by Antibiotic Resistance Plasmid
PLOS ONE | www.plosone.org 10 September 2014 | Volume 9 | Issue 9 | e107716
containing 105 cells were serially diluted to 104, 103,102, 101, and
100 with 900 ml PBS. Each 5 ml of diluted solution was spotted on
a plate and incubated at 30uC for 12 h. Antibiotics were amended
at sub-MIC levels on nutrient agar supplemented with norfloxacin
(Nor, 2 mg/ml), ampicillin (Amp, 25 mg/ml), gentamicin (Gm,
0.2 mg/ml), kanamycin (Km, 0.5 mg/ml), rifampicin (Rif, 4 mg/
ml), or chloramphenicol (32 mg/ml).
8. Bacterial inhibition assay to determine sensitivity tooxidative stress
A bacterial inhibition assay was conducted to determine the
sensitivity of Acinetobacter strains to oxidative stress. Bacterial cell
growth was monitored by measuring the OD600 of the cultures
using a Biophotometer (Eppendorf, Germany). When the bacterial
cell cultures reached the exponential phase (OD600 of ,0.4), 5 ml
of washed cell culture (with PBS) was mixed with 25 ml of
autoclaved nutrient agar. Autoclaved paper disks (860.7 mm,
ADVANTEC, Japan) were placed on the plate and 25 ml of each
oxidative compound was added to a final concentration of 1 M
hydroperoxide (H2O2), 1 M cumene hydroperoxide (CHP),
100 mM menadione (MD), and 100 mM paraquat (PQ). After
incubation at 30uC for 24 h, the clear zones were evaluated by
measuring their diameter (mm). All quantitative data were
obtained from two independent cultures.
9. Swimming and swarming motilitySwimming and swarming motility were tested on 0.2% and
0.5% agar-containing medium, respectively. A. oleivorans DR1
strains were grown to the exponential growth phase (OD600 of
,0.4) and 1 ml of ,109 cell culture was spotted onto nutrient
media. After 24 h and 72 h incubation at 30uC, bacterial motilities
were evaluated by measuring their diameter (mm). All quantitative
data were obtained in triplicate.
10. Cell membrane permeability assaysA fluorescent probe, 8-anilino-1-naphthylenesulfonic acid
(ANS; Sigma-Aldrich, USA), was used to assess the integrity of
bacterial cell membranes. Overnight cultures were diluted 100 fold
in 5 ml fresh medium and grown to the logarithmic growth phase
at 30uC with shaking at 220 rpm. After the cells were treated with
or without 0.5 mg/ml TC at the exponential phase (OD600 = ,0.4)
for 30 min, a 1-ml cell culture was harvested by centrifugation
(13,0006g, 1 min) and washed twice with PBS. The resuspended
solutions were supplemented with ANS (1 ml, 3 mM) at room
temperature for 10 min in the dark. The fluorescence intensity of
cells was quantified using a microplate reader. The filter set for
fluorescence included a 555 nm excitation filter and 590 nm
emission filter. The possibility of different growth rates under the
experimental conditions was excluded by normalizing mg protein.
Cell membrane permeability assays were performed three times
independently. Statistical significance of differences was calculated
using Student’s t-test.
11. Transmission electron microscopyOvernight cultures were diluted 100 fold in 5 ml fresh medium
and grown to the logarithmic growth phase at 30uC with shaking
at 220 rpm. A 1-ml cell culture was harvested by centrifugation
(10,0006g, 1 min) and was washed twice with PBS. Each sample
was fixed with Karnovsky’s fixative containing 0.5% calcium
chloride (Sigma-Aldrich, USA), 2% glutaraldehyde (MERCK,
Germany), and 2% paraformaldehyde (MERCK, Germany) in
0.1 M phosphate buffer (pH 7.4) for 2 h and washed three times.
Samples were post-fixed with 1% osmium tetroxide (OsO4;
Polysciences, USA) dissolved in 0.1 M PB for 2 h and dehydrated
in a gradually ascending ethanol series (50–100%) and infiltrated
with propylene oxide. Specimens were embedded using a Poly/
Bed 812 kit (Polysciences, USA). After embedding in pure fresh
resin and polymerization at 60uC in an electron microscope oven
(TD-700, DOSAKA, Japan) for 24 h, 350-nm-thick sections were
cut and stained with toluidine blue for light microscopy. Seventy-
nanometer-thick sections were double stained with 7% (20 min)
uranyl acetate and lead citrate for contrast staining. Sections were
cut using a LEICA EM UC-7 Ultra-microtome (Leica Micro-
systems, Austria). All the thin sections were observed using a
transmission electron microscope (JEM-1011, 80Kv JEOL, Japan)
at an acceleration voltage of 80 kV.
12. Growth competition assayA competition assay between the wild-type and plasmid-
harboring strains was performed to determine growth fitness; cells
were grown in nutrient media with or without tetracycline
(0.5 mg/ml), hydrogen peroxide (0.15 mM), and norfloxacin
(2 mg/ml) or MSB media supplemented with 2% n-hexadecane.
Two exponentially grown cell cultures (OD600 of ,0.4) were
washed twice with PBS and ,106 CFU/ml of each cell type was
mixed in the appropriate media. After 3 days of cultivation at
30uC with shaking at 220 rpm, relative fitness was determined by
measuring the total number of viable cells on nutrient agar or
selective plates containing TC for wild-type DR1 or DR1
(pAST2), respectively. The relative fitness was calculated using
the following equation [Colony number of antibiotics variants/
(Total viable cells - colony number of antibiotics variant)].
Quantitative data were obtained from two independent mixed
cultures.
Supporting Information
Figure S1 An xy plot of RPKM values from DR1 strainsgrown on nutrient with or without TC (MIC, 1 mg/ml). A
dot indicates a gene, and its x and y coordinates indicate the
RPKM from the following data sets. (A) DR1 (pAST2)/DR1; (B)
DR1 (pAST2)-TC/DR1-TC; (C) DR1 (pAST2)-TC/DR1
(pAST2); (D) DR1-TC/DR1. Fold changes (RPKM ratio) are
represented with a color gradient. Red dots indicate genes
upregulated more than 2 fold. Levels of gene expression (22,
fold change values,2) are shown in three different colors. Orange
dots indicate 1.5#fold change values of,2, gray dots show 21.5,
fold change values of,1.5, and blue dots indicate 22,fold
change values of#21.5. Dark blue dots indicate genes downreg-
ulated less than 2 fold.
(TIF)
Figure S2 Clusters of orthologous groups (COGs)assignments of differently expressed genes. The number
of upregulated and downregulated genes was sorted according to
COGs. Colors of the bars indicate the fold change in gene
expression. Red, gene expression with more than a 2-fold change
in value; Gray, gene expression with between a 22 and 2-fold
change in value; Blue, gene expression with less than a 22-fold
change in value. One-letter abbreviations for functional categories:
A, RNA processing and modification; C, energy production and
conversion; D, cell cycle control and mitosis; E, amino acid
metabolism and transport; F, nucleotide metabolism and trans-
port; G, carbohydrate metabolism and transport; H, coenzyme
metabolism; I, lipid metabolism; J, translation, including ribosome
structure and biogenesis; K, transcription; L, replication, recom-
bination, and repair; M, cell wall structure, biogenesis, and outer
membrane; N, secretion, motility, and chemotaxis; O, molecular
Alteration of Host Phenotypes by Antibiotic Resistance Plasmid
PLOS ONE | www.plosone.org 11 September 2014 | Volume 9 | Issue 9 | e107716
chaperones and related functions; P, inorganic ion transport and
metabolism; Q, secondary metabolite biosynthesis, transport, and
catabolism; T, signal transduction; U, intracellular trafficking,
secretion, and vesicular transport; and V, defense mechanisms. (A)
DR1 (pAST2)/DR1; (B) DR1 (pAST2)-TC/DR1-TC; (C) DR1
(pAST2)-TC/DR1 (pAST2); (D) DR1-TC/DR1.
(TIF)
Figure S3 Sensitivity of DR1 strains to six antibioticswith or without TC supplementation. (A) Sensitivity of DR1
strains to six antibiotics in control nutrient media. (B) Sensitivity of
DR1 strains to six antibiotics with sub-MIC levels of TC (0.5 mg/
ml). Antibiotics were amended at sub-MIC levels. Nutrient media
was supplemented with norfloxacin (Nor, 2 mg/ml), ampicillin
(Amp, 25 mg/ml), gentamicin (Gm, 0.2 mg/ml), kanamycin (Km,
0.5 mg/ml), rifampicin (Rif, 4 mg/ml), or chloramphenicol (32 mg/
ml).
(TIF)
Figure S4 Confirmation of gene expression data fromRNA-Seq using qRT-PCR. Ten genes were selected based on
category and expression value. Error bar of qRT-PCR data was
obtained from triplicate experiments (A) Fold change of
DR1(pAST2)/DR1 (B) Fold change of DR1(pAST2)-TC/DR1-
TC.
(TIF)
Figure S5 Construction of tetH efflux pump mutantstrain, DR1 (pAST2DPtetH). (A) tetH-tetR gene disruption
strategy. Deletion of the intergenic region between tetH and tetRwas performed according to the following method. First, a partial
tetH gene (463 bp) was cloned into the ampicillin-marked shuttle
vector. Second, internal tetR (440 bp) was inserted into the
constructed vector near the tetH gene. Third, a full-length
kanamycin cassette (1264 bp) from the plasmid pUC4K was
inserted between tetH and tetR. The cloned fragment, tetH-
tetR::km, was inserted into the suicide vector and finally
transformed into DR1 (pAST2). (B) PCR verification of recom-
binant strains using the tetH-F/tetR-F primer pair. M, 1-kb DNA
ladder (Fermentas); 1, E. coli Top10 control; 2, DR1 wild-type; 3,
E. coli Top10 (pBBR1MCS4-tetHtetR), 903 bp; 4, DR1 (pAST2),
1326 bp; 5, E. coli Top10 (pBBR1MCS4-tetHtetR::km), 2167 bp;
6, DR1 (pAST2DPtetH), 2167 bp. (C) PCR verification of mutant
plasmid (pAST2DPtetH) construction using the tetH-F/tetR-F
primer pair. M, 1-kb DNA ladder (Fermentas); 1, plasmid DNA
of pAST2 (1326 bp); 2, plasmid DNA of pCVD442-tetHtetR::km(2167 bp); 3, plasmid DNA of pAST2DPtetH (2167 bp).
(TIF)
Figure S6 Standard curves were constructed usingrelative light units (RLU) and known ATP concentra-tions.
(TIF)
Table S1 Antibiotic resistance and oxidative stress-related gene expression profiles.
(DOC)
Table S2 Outer membrane-related gene expressionprofiles.
(DOC)
Table S3 Bacterial strains, plasmids, and primers usedin this study.
(DOC)
Author Contributions
Conceived and designed the experiments: HH JJ WP. Performed the
experiments: HH JJ. Analyzed the data: HH JJ WP. Contributed reagents/
materials/analysis tools: HH JJ WP. Contributed to the writing of the
manuscript: HH WP.
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Alteration of Host Phenotypes by Antibiotic Resistance Plasmid
PLOS ONE | www.plosone.org 13 September 2014 | Volume 9 | Issue 9 | e107716